Development of a “standard” urine specimen

The aim of this initiative is the establishment of a well characterized standard urine sample that will be available to all laboratories working in the field of urinary proteomics to assess platform capability (assessment of pre-analytical steps, platform performance, normalisation), and also enable comparison of datasets.

As a start in this direction, urine was collected from 7 male and 8 female apparently healthy volunteers (aged 20-50). All samples were midstream urine, collected between 8 and 10 am and frozen immediately according to the standard protocol for urine collection. Samples were subsequently thawed and pooled for the generation of two standards (male and female pools) at 1, 10 and 50ml aliquots (http://www.eurokup.org/node/127).

In a multi-institutional study the proteome and peptidome of these samples were analysed using all state-of the art technologies available to us: CE-MS, LC-MS, LC-MS/MS, GeLC-MS, and 2DE-MS. In several cases analysis involved a definition of the actual biochemical entities, i.e. proteins/peptides associated with molecular mass and detected posttranslational modifications and the relative abundance of these compounds. In addition, conventional analytical values from immunological assays and clinical laboratory testing were obtained. In all cases, participating laboratories applied their established protocols for sample preparation, analysis, and data processing.

Data quality was assessed based on intralab and in some cases (CE-MS) inter-lab reproducibility of the data as well as their similarity to published urinary data for each technology. The results of this analysis support that while the samples have not been analyzed “to completion”, i.e., not all of their compounds are currently known and quantified, they are the by far most extensively and comprehensively investigated urine samples reported on to date; more importantly, they are available to all urine proteomics researchers upon request, forming thereby the basis for the comprehensive compilation of the urine proteome.